The kinetochore mediates the movement of chromosomes along the microtubules of the mitotic spindle. The Cyert antibody, which is directed to an incompletely characterized, phosphorylated epitope, binds to the kinetochores of chromosomes in prometaphase in a unprecedented and intriguing manner. Chromosomes moving toward the metaphase plate express the phospho-epitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This differential expression of the epitope may reflect a biochemical pathway regulating chromosome movement and the progression of mitosis. Two competing hypotheses for the molecular nature of the epitope recognized by the Cyert antibody will be tested. First, the antibody recognizes a regulatory phosphorylation on a protein that powers or guides chromosome movement. Second, the epitope recognizes an enzymatic intermediate of a phosphatase that is differentially regulated at kinetochores. The aims of this application are: (1) to examine how expression of the phospho-epitope changes in relation to chromosome movement and mitotic progression; (2) to determine how kinases and phosphatases regulate that expression; (3) to identify the proteins that contain the epitope; and (4) to understand how positional and mechanical signals in the mitotic spindle are converted to biochemical information that regulates chromosome movement and progression through mitosis. These aims will be achieved by immunochemical isolation, fractionation, and molecular characterization of kinetochore proteins. These efforts will be complemented by the analysis of chromosome behavior in live cells, immunolabeling studies, micromanipulation of chromosomes, and the microinjection of antibodies and inhibitors.